Project Summary/Abstract Histone modification and cytosine-5 DNA methylation represent two important epigenetic marks. These two very distinct modifications work in close cooperation to control transcriptional potential, thereby influencing a wide diversity of biological processes. The past decade has seen an explosion of interest in the role of these epigenetic modifications in human heath and disease. The goal of the proposed application is to advance our understanding of these two modifications, by developing and applying two novel in vivo imaging technologies capable of visualizing the epigenetic and even genetic consequences of these modifications. The first specific aim is to develop an in vivo imaging system to assess the causal influence of individual epigenetic modifications. The technology is designed to both achieve selective introduction of a desired modification at a predetermined genomic locus, and to produce a quantitative optical readout of the effect of the epigenetic modification on transcription. This is achieved through the sequence-specific recruitment of epigenetic modifier proteins to a promoter driving a fluorescence-luminescence fusion reporter. The power of this approach comes from the combination of the quantitative optical analysis and the localized recruitment. This will allow high-throughput evaluation of the primary causal effects of individual epigenetic modifications. The second specific aim is to develop an imaging assay for the in vivo visualization of CpG transition mutations. Transition mutations at the epigenetic DNA methylation mark are responsible for approximately one-third of all human hereditary disease mutations and for nearly 50% of all p53 point mutations found in human colorectal cancer. However, the lack of imaging tools for this type of epigenetically induced mutagenesis has held back our understanding of the timing and cell-type specificity of this event in vivo. The proposed system is designed to provide direct visualization of the result of the mutation event. This is achieved through the use of a mutant green fluorescent protein that produces a fluorescent signal upon CpG transition mutation in the chromophore region. The conversion of a specific genetic mutation into an optical signal provides an attractive opportunity to analyze the mutation without employing direct sequencing. The in situ analysis of mutagenesis would allow us to not only determine tissue-specific and cell-type specific in vivo CpG mutation frequencies, but also to analyze mutation kinetics.